Recombinant microorganisms for increased production of organic acids

ABSTRACT

Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant  Actinobacillus succinogenes  that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

STATEMENT REGARDING U.S. GOVERNMENT SUPPORT

This invention was made with support from the United States Governmentunder Cooperative Agreement No. DE-FC36-02GO12001 awarded by theDepartment of Energy. The United States Government has certain rights inthis invention.

This application is a continuation of U.S. non-provisional applicationSer. No. 11/722,579, filed on Aug. 28, 2008, now U.S. Pat. No.8,119,377, which is a U.S. national stage application of PCTInternational Application No. PCT/US2005/045714, filed Dec. 16, 2005,which claims priority from U.S. provisional application Ser. No.60/647,141, filed on Jan. 26, 2005; and U.S. provisional application No.60/639,443, filed on Dec. 22, 2004. The aforementioned applications areincorporated herein by reference in their entireties.

This application contains a Sequence Listing submitted in ASCII formatvia EFS-Web, created on Mar. 9, 2012, named 35548-67.txt, 16,429 bytesin size, and which is incorporated by reference in its entirety.

BACKGROUND

Many chemicals that are currently derived from petrochemical materialscould be produced from naturally occurring carbohydrates. In particular,succinic acid, a four-carbon dicarboxylic acid, has the potential tobecome a high volume commodity chemical that could be used as startingmaterial for commercial processes that produce many importantintermediate and specialty chemicals for the consumer product industriesand that currently rely on starting materials derived from non-renewablepetrochemical materials. For example, as a commodity chemical, succinicacid could replace petrochemical starting materials used in theproduction of 1,4-butanediol (BDO) and tetrahydrofuran (THF) compounds,which are useful as solvents and starting materials for many industries.For example, BDO and THF compounds are useful for producing resins forautomotive bodies, thermoplastics for use in household appliances, andelastic polymers such as Lycra™ in the textile industry. In addition,BDO and THF compounds also have many specialty uses in the agrochemicaland pharmaceutical industries. Notably, worldwide consumption of BDO isexpected to increase at an annual rate as high as 4%.

The petrochemicals currently used to produce BDO and THF includeacetylene, formaldehyde, butane, butadiene, and propylene oxide. All ofthese have various hazardous properties, such as extreme flammability,chemical instability and toxicity. Further, as these materials arederived from petroleum, they deplete a non-renewable resource, and upondisposal or destruction, ultimately release carbon (as carbon dioxide)into the atmosphere. Thus, developing succinic acid as a replacement forpetrochemically derived materials would reduce handling and storage ofhazardous materials, enhance industrial and community safety, reducepollution and environmental costs, and reduce dependence on oil.

Production of succinic acid and other organic compounds by fermentationof sugars is economically feasible. A number of microorganisms have beenused to produce succinic acid using corn sugars as a carbon source. Assuch, developing succinic acid as replacement for petrochemical startingmaterials would expand markets for corn, and other agricultural productsand/or biomass that can provide fermentable sugars.

Formally, the biochemical pathway for succinic acid production adds acarbon dioxide molecule to the three carbon compound phosphoenolpyruvate(PEP), to produce the four carbon compound oxaloacetate (OAA). The nextsteps in the pathway to succinic acid are part of the reversetricarboxylic acid cycle (TCA cycle) and include two obligate reductionsteps. In the biochemical process leading from OAA to succinate, OAAmust first be reduced to produce L-malate. L-malate is then dehydratedto produce fumarate and water. Fumarate is then reduced to give thesuccinic acid. In the chemical arts, “reduction” refers to the additionof molecular hydrogen to a compound.

Generally, free molecular hydrogen is not found in intracellularbiological systems. Rather, reduction is performed through the use ofcoenzymes that function as biochemical equivalents of hydrogen (i.e., ascarriers of molecular hydrogen) and are termed “reducing equivalents.”Reducing equivalents include the coenzymes nicotinamide adeninedinucleotide hydrogen (“NADH”), nicotinamide adenine dinucleotidephosphate hydrogen (“NADPH”), flavine adenine dinucleotide hydrogen(“FADH₂”), and flavin mononucleotide hydrogen (“FMNH”). Generally, NADHand NADPH may be interconverted in a range of microorganisms by theenzyme pyridine dinucleotide transhydrogenase.

the reducing equivalents required to transform OAA to succinate areprovided by NAD(P)H₂, FADH₂, or other co-factors. It is essential that asufficient quantity of reducing equivalents is available for thetransformation of OAA to succinate. If sufficient reducing equivalentsare not available, the biochemical pathway will not functionefficiently, and only a portion of the OAA will be transformed into thedesired succinate.

Reducing equivalents may be produced in a number of biological processesthat are commonly found in cellular metabolism. For example, reducingequivalents may be generated in the pentose phosphate cycle (PPC). Inthe PPC, glucose-6-phosphate is converted toD-6-phospho-glucono-δ-lactone by the enzyme glucose-6-phosphatedehydrogenase, which is also known as Zwischenferment enzyme or Zwf. Aspart of this conversion, NADP is converted to NADPH as an acceptor ofreducing equivalents.

Few microorganisms have been described which produce sufficientconcentrations of succinic acid for commercial production. One suchmicroorganism is Actinobacillus succinogenes, a facultative anaerobethat was isolated from the bovine rumen. This organism produces highconcentration of succinic acid and tolerates high sugar concentration.Actinobacillus succinogenes is one of the best known producers ofsuccinic acid, but the fermentative yields of this strain may be limitedby the lack of reducing equivalents. As such, improvements are desirableto increase the yield of succinic acid produced by fermentation,including the use of improved strains of microorganisms for producingsuccinic acid.

SUMMARY

Disclosed are recombinant microorganisms for producing organic acids.The recombinant microorganisms expresses a polypeptide that has one ormore biochemical activities of an enzyme utilized in the pentosephosphate cycle. In one embodiment, the enzyme isglucose-6-phosphate-1-dehydrogenase, also called Zwischenferment enzymeor Zwf. For example, the recombinant microorganism may express apolynucleotide that encodes a polypeptide having Zwf enzyme activity. Inone embodiment, the recombinant microorganism is a recombinant strain ofa succinic acid producing microorganism which has been transformed witha DNA molecule that expresses a polypeptide having Zwf enzyme activity.

The recombinant microorganism typically is capable of producing one ormore organic acids at a level suitable for commercial production. Insome embodiments, the recombinant microorganism is a succinic acidproducing microorganism. For example, the microorganism may producesuccinic acid at a concentration suitable for commercial production. Aconcentration suitable for commercial production may be at least about20 g/L, 40 g/L, 60 g/L, 80 g/L, 100 g/L, 120 g/L, and/or 140 g/L.Desirably, the recombinant microorganism is capable of producingsuccinic acid at concentrations of about 50 g/L to about 130 g/L.

The recombinant microorganism may be selected and/or recombinantlyengineered to tolerate relatively high concentrations of succinic acidto facilitate production of succinic acid at a concentration suitablefor commercial production in a fermentation system. In some embodiments,the recombinant microorganism may be selected to produce relatively lowamounts of undesirable by-products such as acetate, formate, and/orpyruvate (e.g., no more than about 2.0 g/L acetate, no more than about2.0 g/L formate, and/or no more than about 3.0 g/L pyruvate). Therecombinant microorganism may be derived from a strain (or a variant ofa strain) that is resistant to levels of sodium monofluoroacetate atconcentration of at least about 1 g/L, 2 g/L, 4 g/L, and/or 8 g/L. Inanother embodiment, a variant of the recombinant microorganism may beselected to be resistant to levels of sodium monofluoroacetate atconcentration of at least about 1 g/L, 2 g/L, 4 g/L, and/or 8 g/L.

In one embodiment, the recombinant microorganism is derived from astrain of Actinobacillus succinogenes (i.e., “A. succinogenes”) or amicroorganism related to Actinobacillus succinogenes. One suitablestrain of A. succinogenes is Bacterium 130Z deposited with the AmericanType Culture Collection (ATCC), under ATCC Accession Number 55618. SeeU.S. Pat. No. 5,504,004 for description of Bacterium 130Z and othersuitable strains.

Other suitable microorganisms may be selected for preparing therecombinant microorganism and may include microorganisms which arerelated to A. succinogenes as determined by sequence identity within 16SrRNA. For example, a suitable microorganism related to A. succinogenesmay have 16S rRNA that exhibits substantial sequence identity to A.succinogenes 16S rRNA (i.e., a microorganism having 16S rRNA thatexhibits at least about 90% sequence identity to A. succinogenes 16SrRNA or more suitably, that exhibits at least about 95% sequenceidentity to A. succinogenes 16S rRNA). Many representativemicroorganisms of the family Pasteurellaceae have 16S rRNA that exhibitsat least about 90% sequence identity to A. succinogenes 16S rRNA. Forexample, See Guettler et al., INT'L J. SYSTEMATIC BACT. (1999), 49,207-216 at page 209, Table 2. Suitable microorganisms may includemicroorganisms such as Bisgaard Taxon 6 and Bisgaard Taxon 10.

In some embodiments, the recombinant microorganism may be prepared fromorganisms other than A. succinogenes. For example, the recombinantmicroorganism may be prepared from any microorganism that is suitablefor use in fermentation systems for producing organic acids. A suitablemicroorganism may include E. coli. Suitable strains of E. coli are knownin the art.

Variants of microorganisms that are resistant to sodiummonofluoroacetate may also be suitable for preparing the recombinantmicroorganism. For example, See U.S. Pat. Nos. 5,521,075 and 5,573,931.In one embodiment, the recombinant microorganism is prepared from avariant of A. succinogenes that is resistant to at least about 1 g/Lsodium monofluoroacetate. One suitable variant is FZ45. See U.S. Pat.No. 5,573,931. The recombinant microorganism deposited under ATCCAccession Number PTA-6255, is derived from a variant of A. succinogenesthat is resistant to at least about 1 g/L sodium monofluoroacetate(i.e., FZ45).

The recombinant microorganism typically is transformed with apolynucleotide encoding a polypeptide that has one or more biochemicalactivities of an enzyme utilized in the pentose phosphate cycle. Forexample, the recombinant microorganism may be transformed with apolynucleotide that encodes a polypeptide having one or more biochemicalactivities of the Zwf enzyme (i.e., glucose-6-phosphate dehydrogenaseactivity and/or NADP reductase activity). Desirably, the polynucleotideencodes a polypeptide that facilitates the conversion of NADP to NADPH.The polynucleotide or polypeptide may be endogenous to the microorganismor derived from a gene or enzyme normally present in the microorganism.In some embodiments, the polynucleotide or polypeptide may be homologousto an endogenous gene or enzyme of the microorganism. In otherembodiments, the polynucleotide or polypeptide may be heterologous(i.e., derived from a gene or enzyme normally not present in themicroorganism or derived from a source other than the microorganism).

The recombinant microorganism may express a variant of thepolynucleotide that encodes the polypeptide and/or a variant of thepolypeptide. A variant of the polynucleotide may include apolynucleotide having at least about 90% sequence identity to thepolynucleotide, or desirably, at least about 95% sequence identity tothe polynucleotide, where the polynucleotide encodes a polypeptide thathas one or more biochemical activities of the Zwf enzyme (e.g., NADPreductase activity). A variant may include a polypeptide that has atleast about 90% sequence identity to the polypeptide, or desirably, atleast about 95% sequence identity to the polypeptide, where thepolypeptide has one or more biochemical activities of the Zwf enzyme(e.g., NADP reductase activity). As such, suitable polynucleotides mayinclude polynucleotides encoding a polypeptide having at least about 95%sequence identity to a selected Zwf enzyme, where the polypeptide hasNADP reductase activity.

The recombinant microorganism may be transformed with a polynucleotidethat expresses a polypeptide having Zwf enzyme activity, where therecombinant microorganism exhibits higher Zwf enzyme activity than amicroorganism which has not been transformed with a polynucleotide thatexpresses a polypeptide having Zwf enzyme activity. In some embodiments,the recombinant microorganism exhibits at least about five times (5×)more Zwf enzyme activity, (or desirably at least about ten times (10×)more Zwf enzyme activity, or more desirably at least about fifty times(50×) more Zwf enzyme activity), than a microorganism which has not beentransformed with a polynucleotide that expresses a polypeptide havingZwf enzyme activity. Zwf enzyme activity may include NADP reductaseactivity. Zwf enzyme activity may be determined by measuring the levelof NADPH present the recombinant microorganism (e.g., as compared to amicroorganism which has not been transformed with a polynucleotide thatexpresses a polypeptide having Zwf enzyme activity).

The recombinant microorganism may express a polynucleotide that encodesa Zwf enzyme such as a Zwf gene. A variant of the polynucleotide maycomprise a polynucleotide having at least about 90% sequence identity toa Zwf gene, or desirably, at least about 95% sequence identity to a Zwfgene and encoding a polypeptide that has one or more biochemicalactivities of the Zwf enzyme. A variant of a polynucleotide may includea nucleic acid fragment of the polynucleotide. For example, a fragmentmay include at least about 90% of a Zwf gene, or at least about 95% of aZwf gene. A nucleic acid fragment may be any suitable length. Forexample, the nucleic acid fragment may comprise at least about 10, 50,100, 250, 500, 1000 and/or 1400 nucleotides. A fragment may encode apolypeptide that has one or more biochemical activities of the Zwfenzyme.

Suitable Zwf genes may include Zwf genes endogenous or native to therecombinant microorganism (i.e., Zwf genes normally present in themicroorganism from which the recombinant microorganism is derived), orvariants thereof. Other suitable Zwf genes may include Zwf genesheterologous to the microorganism (i.e., Zwf genes normally not presentin, or obtained from sources other than the microorganism used toprepare the recombinant microorganism), or variants thereof. SuitableZwf genes may include variants that have at least about 90% sequenceidentity to the polynucleotide sequence of the selected Zwf gene(preferably at least about 95% sequence identity to the polynucleotidesequence of the selected Zwf gene) and that encode a polypeptide thathas one or more biochemical activities of the Zwf enzyme (i.e.,glucose-6-phosphate dehydrogenase activity and/or NADP reductaseactivity).

Suitable Zwf genes may include the E. coli Zwf gene or variants thereof.The polynucleotide sequence of the E. coli Zwf gene is deposited withGenBank under accession number NC_(—)000913, reverse complement ofnucleotides 1,932,863 to 1,934,338 (SEQ ID NO:1) and under accessionnumber M55005, nucleotides 708 to 2180 (SEQ ID NO:2). Suitable variantsof the E. coli Zwf gene may include a polynucleotide having at leastabout 90% sequence identity (desirably at least about 95% sequenceidentity) to the polynucleotide of SEQ ID NO:1 (or SEQ ID NO:2), suchthat the polynucleotide encodes a polypeptide that has one or morebiochemical activities of the Zwf enzyme (i.e., glucose-6-phosphatedehydrogenase activity and/or NADP reductase activity).

Suitable Zwf genes may include the A. succinogenes Zwf gene or variantsthereof. The draft genome sequence for A. succinogenes 130Z has recentlybeen established and assembled and is publicly available as of September2005, at the Joint Genome Institute, Department of Energy website. TheZwf gene is annotated as “glucose-6-phosphate 1-dehydrogenase” and ispresent on contig 115, nucleotides 8738-10225 (i.e., SEQ ID NO:5). Thepredicted amino acid sequence of encoded polypeptide (i.e., the A.succinogenes Zwf enzyme) is presented as SEQ ID NO:6. The Zwf enzymeexhibits 43% amino acid sequence identity and 60% amino acide homologyto the E. coli Zwf enzyme using the “BLAST” alignment algorithm versionBLASTP 2.2.12, BLOSUM62 matrix, available at the National Center forBiotechnology Information website. Suitable variants of the A.succinogenes Zwf gene may include a polynucleotide having at least about90% sequence identity (desirably at least about 95% sequence identity)to the polynucleotide of SEQ ID NO:5, such that the polynucleotideencodes a polypeptide that has one or more biochemical activities of theZwf enzyme (i.e., glucose-6-phosphate dehydrogenase activity and/or NADPreductase activity).

The recombinant microorganism may express an endogenous Zwf enzyme(i.e., a Zwf enzyme present within the microorganism from which therecombinant microorganism is derived), or variants thereof. In otherembodiments, the recombinant microorganism may express a Zwf enzyme thatis heterologous to the microorganism (i.e., a Zwf enzyme that is notpresent or expressed in the microorganism from which the recombinantmicroorganism is derived), or variants thereof. Suitable Zwf enzymes mayinclude variants having at least about 90% amino acid sequence identityto the amino acid sequence of a selected Zwf enzyme (desirably at leastabout 95% amino acid sequence identity to the selected Zwf enzyme) andhaving one or more biochemical activities of the Zwf enzyme (e.g., NADPreductase activity and/or glucose-6-phosphate dehydrogenase activity).Suitable Zwf enzymes may include the E. coli Zwf enzyme (e.g., SEQ IDNO:3, polypeptide encoded by the reverse complement of the nucleotidesequence of nucleotides 1,932,863 to 1,934,338 of NC_(—)000913) orvariants thereof, and the A. succinogenes Zwf enzyme (e.g., SEQ ID NO:6)or variants thereof.

A variant polypeptide may include a fragment of a Zwf enzyme. Forexample, a fragment may include at least about 90% of the amino acidsequence of SEQ ID NO:3, or more desirably at least about 95% of theamino acid sequence of SEQ ID NO:3. In other embodiments, a fragment mayinclude at least about 90% of the amino acid sequence of SEQ ID NO:6, ormore desirably at least about 95% of the amino acid sequence of SEQ IDNO:6. A polypeptide fragment may be any suitable length. For example,the polypeptide fragment may comprise at least about 10, 50, 100, 200,and/or 300 amino acids (e.g., of SEQ ID NO:3 or SEQ ID NO:6). Apolypeptide fragment typically has one or more biochemical activities ofthe Zwf enzyme.

The recombinant microorganism may include a succinic acid producingmicroorganism that has been transformed with a polynucleotide thatexpresses an endogenous (i.e., native) Zwf gene which encodes anendogenous (i.e., native) Zwf enzyme. In some embodiments, therecombinant microorganism may include a succinic acid producingmicroorganism that has been transformed with a polynucleotide thatexpresses a heterologous Zwf gene which encodes a heterologous Zwfenzyme. The recombinant microorganism deposited with the American TypeCulture Collection (ATCC), under ATCC Accession Number PTA-6255, is arecombinant strain of a succinic acid producing microorganism (i.e., A.succinogenes) that expresses a heterologous Zwf gene (e.g., the E. coliZwf gene) which encodes a heterologous Zwf enzyme.

The recombinant microorganism may express a polypeptide having Zwfenzyme activity at relatively high levels (i.e., the polypeptide may be“overexpressed”). For example, the recombinant microorganism may expressan endogenous Zwf enzyme at relatively high levels as compared to anon-recombinant microorganism. In some embodiments, the recombinantmicroorganism may be transformed with a DNA molecule (e.g., a plasmid)that expresses an endogenous Zwf enzyme at relatively high levelscompared to a recombinant microorganism that has not been transformedwith the DNA molecule.

A polynucleotide, such as a Zwf gene, may be optimized for expression ina selected microorganism from which the recombinant microorganism isderived. For example, a heterologous Zwf gene may be optimized forexpression in a non-native microorganism. In some embodiments, a Zwfgene may be optimized for expression in A. succinogenes, or in amicroorganism such as Bisgaard Taxon 6 or Bisgaard Taxon 10. In otherembodiments, a Zwf gene may be optimized for expression in E. coli.

A polynucleotide such as a Zwf gene may be optimized for expression inthe recombinant microorganism by any suitable strategy. For example, aZwf gene may be optimized for expression in the recombinantmicroorganism by operably linking the Zwf gene to a promoter sequencethat facilitates expression of the Zwf gene in the recombinantmicroorganism. The promoter sequence may be optimized to facilitaterelatively high levels of expression in the recombinant microorganism(i.e., optimized to facilitate “overexpression”). The Zwf gene may beoperably linked to a promoter sequence that is endogenous to themicroorganism (i.e., a promoter native to the microorganism) orheterologous to the microorganism (i.e., a promoter normally not presentin, or derived from a source other than the microorganism). Suitablepromoters may include promoters that are not the native promoter for theselected Zwf gene (i.e., a non-Zwf gene promoter, which may beendogenous to the microorganism or heterologous to the microorganism).Suitable promoters may include inducible promoters or constitutivepromoters. Suitable promoters may be derived from promoters of succinicacid producing microorganisms.

In other embodiments, expression of a Zwf gene may be optimized at thetranslational level. For example, a heterologous Zwf gene may bemodified to include codons that demonstrate preferred usage frequency inthe microorganism from which the recombinant microorganism is derived asa non-natural host for the gene.

In another embodiment, expression of a polynucleotide such as a Zwf genemay be optimized by providing a relatively high copy number of thepolynucleotide in the recombinant microorganism. For example, a Zwf genemay be present on an epigenetic element that is capable of replicatingto achieve a relatively high copy number in the recombinantmicroorganism (e.g., a plasmid).

In some embodiments, the recombinant microorganism is a recombinantstrain of a succinic acid producing microorganism, such asActinobacillus succinogenes or related microorganisms, which has beentransformed with a DNA molecule that includes a promoter operationallylinked to a Zwf gene. The Zwf gene may be derived from an endogenous orheterologous Zwf gene and may include, for example, the A. succinogenesZwf gene (e.g., SEQ ID NO:5) and the E. coli Zwf gene (e.g., SEQ ID NOs:1 & 2). Other Zwf genes are known and their polynucleotide sequenceshave been published (See, e.g., GenBank). Suitable endogenous or nativepromoter sequences of succinic acid producing microorganisms mayinclude, for example, the phosphoenolpyruvate (PEP) carboxykinasepromoter sequence. The A. succinogenes phosphoenolpyruvate (PEP)carboxykinase promoter sequence is deposited with GenBank underaccession number AY308832, nucleotides 1-258 (SEQ ID NO:4). Aphosphoenolpyruvate (PEP) carboxykinase promoter may be a suitableheterologous promoter for a Zwf gene (i.e., a non-Zwf gene promoter).

As described herein, a recombinant microorganism may include arecombinant DNA molecule as an epigenetic element and/or the recombinantDNA molecule may be incorporated into the genome of the microorganism(e.g., by appropriate methods of recombination). In certain embodiments,the DNA molecule is a plasmid, a recombinant bacteriophage, a bacterialartificial chromosome (3AC) and/or an E. coli P1 artificial chromosome(PAC). The DNA molecule may include a selectable marker. Suitableselectable markers may include markers for kanamycin resistance,ampicillin resistance, tetracycline resistance, chloramphenicolresistance, and combinations of these selectable markers. In oneembodiment, the selectable marker is kanamycin resistance.

As described herein, a recombinant DNA molecule may include a suitablepromoter operationally linked to a polynucleotide that encodes apolypeptide having one or more biochemical activities of Zwf enzyme forexpressing the polynucleotide in a recombinant microorganism (e.g., A.succinogenes). The promoter may be suitable for expressing thepolypeptide in a succinic acid producing microorganism. In someembodiments, the recombinant DNA molecule includes a phosphoenolpyruvate (PEP) carboxykinase promoter (e.g., a A. succinogenesphosphoenol pyruvate (PEP) carboxykinase promoter) operationally linkedto a Zwf gene or a variant thereof, (which may include a heterologousZwf gene such as an E. coli Zwf gene or an A. succinogenes Zwf gene).For example, the DNA molecule may include nucleotides 1-258 of the DNAsequence deposited under GenBank accession number AY308832 (SEQ ID NO:4)or a variant thereof, operationally linked to the reverse complement ofnucleotides 1,932,863 to 1,934,338 of the DNA sequence deposited underGenBank accession number NC_(—)000913 (SEQ ID NO:1); or operationallylinked to the DNA sequence deposited under GenBank accession numberM55005 (SEQ ID NO:2); or operationally linked to the DNA sequence of SEQID NO:5. In some embodiments, the promoter may include a polynucleotidehaving at least about 95% sequence identity to the polynucleotide of SEQID NO:4 and having promoter activity in the recombinant microorganism.

A recombinant microorganism comprising the recombinant DNA molecule maybe suitable for producing an organic acid (e.g., succinic acid or lacticacid) in a fermentation system. The recombinant microorganism comprisingthe recombinant DNA molecule may produce enhanced levels of an organicacid (e.g., succinic acid or lactic acid) in a fermentation systemrelative to a microorganism that does not comprise the recombinant DNAmolecule.

Also disclosed is a DNA plasmid comprising one or more of theaforementioned recombinant DNA molecules. The DNA plasmid may include aselectable marker. Suitable selectable markers may include one or moreof the genes for ampicillin resistance, streptomycin resistance,kanamycin resistance, tetracycline resistance, chloramphenicolresistance, and sulfonamide resistance, operationally linked to asuitable promoter (e.g., a constitutive promoter). In one embodiment,the DNA plasmid includes the gene for kanamycin resistance.

The DNA plasmid may include sequences required for maintaining and/orreplicating the plasmid in one or more suitable host cells. In oneembodiment, the DNA plasmid is capable of functioning as a shuttlevector between suitable host cells. The DNA plasmid may be capable offunctioning as a shuttle vector between A. succinogenes and E. coli.

Also disclosed is a host cell that includes one or more of theaforementioned DNA molecules. For example, the host cell may comprise aDNA plasmid that includes the DNA molecule. The host cell may besuitable for producing and isolating a DNA plasmid that includes the DNAmolecule.

The host cell may be suitable for producing one or more organic acids ina fermentation system. In some embodiments, the host cell expresses aZwf gene (and subsequently a Zwf enzyme) at a level suitable forenhancing the production or one or more organic acids (e.g., succinicacid or lactic acid) in a fermentation system. In some embodiments, thehost cell may expresses a Zwf gene (and subsequently a Zwf enzyme) at alevel suitable for enhancing the concentration of reducing equivalents(e.g., NADPH) in the host cell. The host cell may comprise a recombinantstrain of A. succinogenes that expresses a Zwf gene (and subsequently aZwf enzyme) at a level suitable for enhancing the concentration ofreducing equivalents (e.g., NADPH) in the strain. Such a strain may besuitable for producing enhanced levels of succinic acid in afermentation system relative to a strain that does not comprise therecombinant DNA molecule.

In some embodiments, the host cell is capable of producing succinic acidat concentrations of at least about 20 g/L, 40 g/L, 60 g/L, 80 g/L, 100g/L, 120 g/L, 140 g/L, and/or 160 g/L (e.g., in a fermentation system).In certain embodiments, the host cell is capable of producing succinicacid at concentrations of at about 50 g/L to about 130 g/L. Desirably,the host cell does not produce selected organic acids other thansuccinic acid at substantial concentrations. Where the host cellproduces organic acids other than succinic acid (e.g., acetic acid,formic acid, pyruvic acid, and mixtures thereof), desirably the organicacids other than succinic acid are produced at concentrations no morethan about 30 g/L, more desirably no more than about 20 g/L, moredesirably no more than about 10 g/L, and even more desirably no morethan about 5 g/L.

The aforementioned recombinant microorganisms may be used in methodsthat include fermenting a nutrient medium to produce one or more organicacids. In some embodiments, the methods may include fermenting anutrient medium with a recombinant microorganism that expresses a Zwfgene (e.g., the E. coli Zwf gene). Organic acids produced by the methodmay include succinic acid and lactic acid. In further embodiments, themethods are suitable for producing succinic acid at concentrations of atleast about 20 g/L, 40 g/L, 60 g/L, 80 g/L, 100 g/L, 120 g/L, and/or 160g/L.

In particular, the methods may include fermenting a nutrient medium witha recombinant strain of A. succinogenes that expresses a Zwf gene (andsubsequently a Zwf enzyme) at a level suitable for enhancing theproduction of an organic acid (e.g., succinic acid). The Zwf gene mayinclude a heterologous Zwf gene. A recombinant strain of A. succinogenesthat expresses a heterologous Zwf gene (i.e., the E. coli Zwf gene) isdeposited under ATCC accession number PTA-6255. In certain embodiments,the recombinant microorganism is a recombinant strain of a microorganismsuch as Bisgaard Taxon 6 or Bisgaard Taxon 10 that expresses a Zwf gene(which may be heterologous) at a level suitable for enhancing theproduction of an organic acid (e.g., succinic acid). Suitablerecombinant microorganisms also include recombinant strains of E. colithat express a Zwf gene (which may be heterologous) at a level suitablefor enhancing the production of an organic acid (e.g., lactic acid).

In the method, it may be desirable to ferment a nutrient medium withrecombinant microorganisms that produce relatively high levels ofselected organic acids, such as succinic acid and/or lactic acid. Assuch, the selected recombinant microorganisms may be resistant to highlevels of organic acids, such as succinic and/or lactic acid. Therecombinant microorganisms may also be selected to produce relativelylow levels of other undesirable by-products. For example, therecombinant microorganism may produce relatively low levels of acetate,formate, pyruvate, and mixtures thereof (e.g., no more than about 2.0g/L, no more than about 2.0 g/L formate, and/or no more than about 3.0g/L pyruvate). The above-described recombinant microorganisms that areresistant to concentrations of sodium monofluoroacetate of about 1 g/L,2 g/L, 4 g/L, and/or 8 g/L are suitable for the method.

In the method, the nutrient medium typically includes a fermentablecarbon source. A fermentable carbon source may be provided by afermentable biomass. In one embodiment, the fermentable carbon source isderived from feedstock, including sugar crops, starch crops, and/orcellulosic crop residues. Generally, the fermentable carbon source is asugar, such as glucose. The fermentable carbon source may also includesugar alcohols. In suitable embodiments, the method results in asuccinic acid yield (g) of at least about 100% relative to glucose (g).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Metabolic flux analysis of A. succinogenes variant FZ45 batchfermentation using glucose.

FIG. 2: Metabolic flux analysis of recombinant A. succinogenesFZ45/pJR762.73 batch fermentation using glucose.

FIG. 3: Zwf enzymatic activities in cell extracts of transformedstrains. Extracts were prepared and assayed for Zwf activity asdescribed below. All strains carrying pJR762.73 showed orders ofmagnitude increases in Zwf activity, which is graphed on a logarithmicscale.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Disclosed herein is a recombinant microorganism which expresses apolypeptide that has one or more biochemical activities of an enzymeutilized in the pentose phosphate cycle. As used herein, “microorganism”includes any suitable single-cell organism such as bacteria, fungi, andyeast. As used herein, “recombinant microorganism” means a microorganismthat has been modified in a manner that results in a non-naturallyoccurring microorganism. A “recombinant microorganism” may include amicroorganism that has been transformed with a DNA molecule (e.g., arecombinant DNA molecule).

A recombinant microorganism may include a microorganism that has beentransformed with a DNA molecule that expresses a polypeptide having oneor more biochemical activities of the Zwf enzyme. The pentose phosphatecycle utilizes several enzymes includingglucose-6-phosphate-1-dehydrogenase, (also called Zwischenferment enzymeor Zwf); 6-phosphogluconolactonase; 6-phosphogluconate dehydrogenase,(also called Gnd); ribose-5-phosphate isomerase A and B; ribulosephosphate 3-epimerase; transketolase I and II; and transaldolase A andB. Of these enzymes, Zwf and Gnd result in the production of twohydrogen equivalents in the form of NADPH.

The recombinant microorganism may express any suitable polypeptide orvariant thereof having one or more biochemical activities of the Zwfenzyme (e.g., glucose-6-phosphate-1-dehydrogenase activity and NADPreductase activity). For example, one suitable Zwf enzyme is the E. coliZwf enzyme or a variant thereof. In some embodiments, the recombinantmicroorganism may express the Zwf enzyme at elevated levels (i.e.,“overexpress” the enzyme) relative to levels present in non-recombinantmicroorganisms.

The recombinant microorganism may express a variant polypeptide havingat least about 90% sequence identity to the amino acid sequence of a Zwfenzyme, and more desirably at least about 95% sequence identity to theamino acid sequence of a Zwf enzyme. In suitable embodiments, therecombinant microorganism may express a variant of a Zwf enzyme that hasat least about 96%, 97%, 98%, or 99% sequence identity to the Zwfenzyme. Desirably, the variant polypeptide has one or more biochemicalactivities of the Zwf enzyme. A variant polypeptide may include afragment of the Zwf enzyme. Suitable Zwf enzymes include A. succinogenesZwf enzyme, E. coli Zwf enzyme, and variants thereof.

The recombinant microorganism may express a polynucleotide encoding apolypeptide having one or more biochemical activities of the Zwf enzymesuch as a Zwf gene or a variant thereof. For example, the recombinantmicroorganism may express a Zwf gene or a variant comprising a DNAsequence that has at least about 90% sequence identity to the Zwf gene,and more desirably at least about 95% sequence identity to the Zwf gene.In suitable embodiments, the recombinant microorganism may express avariant of the Zwf gene comprising a DNA sequence that has at leastabout 96%, 97%, 98%, or 99% sequence identity to the Zwf gene.Desirably, the variant polynucleotide encodes a polypeptide having oneor more biochemical activities of Zwf enzyme. A variant polynucleotidemay include a fragment of the Zwf gene. In some embodiments, therecombinant microorganism may express an A. succinogenes Zwf gene, an E.coli Zwf gene, or a variant thereof.

The recombinant microorganism may be derived from any suitablemicroorganism. Typically, the microorganism is capable of producing anorganic acid at a level suitable for commercial production. As usedherein, an “organic acid” includes at least one carboxylic group. Forexample, “organic acid” includes succinic acid and lactic acid. As usedherein, organic acids may be alternately designated by the organic acidanion or a salt thereof. For example, “succinic acid” may be referred toas “succinate”; “lactic acid” may be referred to as “lactate”; “formicacid” may be referred to as “formate”; and “pyruvic acid” may bereferred to as “pyruvate.”

Suitable microorganisms for preparing recombinant microorganisms asdescribed herein may include, but are not limited to, members of theActinobacillus genus, including A. succinogenes; Bisgaard Taxon 6;Bisgaard Taxon 10; Mannheimia succiniciproducens; E. coli;Anaerobiospirillum succiniciproducens; Ruminobacter amylophilus;Succinivibrio dextrinosolvens; Prevotella ruminicola; Ralstoniaeutropha; and coryneform bacteria (e.g., Coryizebacterium glutamicum,Corynebacterium ammoniagenes, Brevibacterium flavum, Brevibacteriumlactofermentuin, Brevibacterium divaricatum); members of theLactobacillus genus; yeast (e.g., members of the Saccharomyces genus);and any subset thereof. Suitable microorganisms for preparingrecombinant microorganisms as described herein may include succinic acidproducing microorganisms.

The recombinant microorganism typically expresses a Zwf gene, which maybe a heterologous Zwf gene. The Zwf gene may be optimized for expressionin the recombinant microorganism. For example, the Zwf gene may beoperationally linked to a promoter that facilitates overexpression ofthe gene in the recombinant microorganism relative to a non-recombinantmicroorganism. The promoter may be endogenous to the microorganism(i.e., native to the microorganism from which the recombinantmicroorganism is derived) or heterologous to the microorganism (i.e.,not native to the microorganism from which the recombinant microorganismis derived or obtained from a source other than the microorganism). Thepromoter may be endogenous to the Zwf gene or heterologous to the Zwfgene (i.e., a non-Zwf gene promoter). The promoter may facilitateconstitutive and/or inducible expression of the Zwf gene, and/or thepromoter may be modified to facilitate constitutive and/or inducibleexpression of the Zwf gene by suitable methods.

The Zwf gene may be modified to facilitate translation of thecorresponding mRNA. For example, the Zwf gene may be modified to includecodons that are not present in the endogenous or native gene. Thesenon-endogenous codons may be selected to reflect the codon usagefrequency in the recombinant microorganism. Codon usage tables have beendeveloped for many microorganisms and are known in the art. The Zwf genemay be modified to reflect the codon usage frequency for A. succinogenesas provided below:

Exemplary Codon Frequency Usage for Actinobacillus succinogenes.

Source: GenBank Release 144.0 [Nov. 12, 2004]

Triplet [frequency per thousand] UUU [20.4] UCU [1.9] UAU [13.0] UGU[7.4] UUC [29.7] UCC [14.8] UAC [16.7] UGC [3.7] UUA [35.3] UCA [13.0]UAA [1.9] UGA [0.0] UUG [20.4] UCG [5.6] UAG [0.0] UGG [16.7] CUU [13.0]CCU [5.6] CAU [5.6] CGU [20.4] CUC [1.9] CCC [0.0] CAC [7.4] CGC [9.3]CUA [0.0] CCA [3.7] CAA [18.6] CGA [1.9] CUG [5.6] CCG [35.3] CAG [3.7]CGG [0.0]) AUU [27.8] ACU [18.6] AAU [13.0] AGU [7.4] AUC [22.3] ACC[31.5] AAC [39.0] AGC [3.7] AUA [0.0] ACA [5.6] AAA [76.1] AGA [1.9] AUG[20.4] ACG [18.6] AAG [1.9] AGG [0.0] GUU [26.0] GCU [13.0] GAU [33.4]GGU [61.2] GUC [7.4] GCC [13.0] GAC [29.7] GGC [24.1] GUA [11.1] GCA[22.3] GAA [64.9] GGA [0.0] GUG [27.8] GCG [35.3] GAG [5.6] GGG [5.6]

The recombinant microorganism may include a recombinant strain of A.succinogenes that expresses a Zwf gene (e.g., an endogenous Zwf geneand/or a heterologous Zwf gene such as the E. coli Zwf gene). Othersuitable microorganisms for producing recombinant microorganisms includeBisgaard Taxon 6 (deposited with the Culture Collection, University ofGoteborg, Sweden (CCUG), under accession number 15568); Bisgaard Taxon10 (deposited under CCUG accession number 15572); and any suitablestrain of E. coli for which many strains are known in the art. Therecombinant microorganism may be derived from a strain that produceshigh levels of one or more organic acids such as succinic acid andlactic acid, and/or the recombinant microorganism may be selected and/orengineered to produce high or enhanced levels of one or more organicacids such as succinic acid and lactic acid relative to anon-recombinant microorganism.

The recombinant microorganism may be derived from strains that areresistant to relatively high levels of undesirable by-products and/orstrains of microorganisms that produce relatively low levels ofundesirable by-products. Undesirable by-products may include formate (orformic acid), acetate (or acetic acid), and/or pyruvate (or pyruvicacid). Methods for selecting strains that produce low levels of acetateare known in the art. See, e.g., U.S. Pat. Nos. 5,521,075 and 5,573,931,which are incorporated herein by reference. For example, strains ofmicroorganisms that produce relatively low levels of acetate may beselected by growing the microorganisms in the presence of a toxicacetate derivative, such as sodium monofluoroacetate at a concentrationof about 1.0 to about 8.0 g/L. Selected strains may produce relativelylow levels of acetate (e.g., less than about 2.0 g/L), formate (e.g.,less than about 2.0 g/L), and/or pyruvate (e.g., less than about 3.0g/L) in a glucose fermentation. One suitable monofluoroacetate resistantstrain for producing a recombinant microorganism is a strain of A.succinogenes called FZ45, which is a derivative of A. succinogenesdeposited under ATCC accession number 55618. See U.S. Pat. No.5,573,931, which describes suitable methods for preparing microbialstrains that are resistant to monofluoroacetate.

The recombinant microorganism may be selected and/or engineered to beresistant to relatively high levels of undesirable by-products and/or toproduce relatively low levels of undesirable by-products. For example,after transformation, a population of recombinant microorganisms may begrown in the presence of sodium monofluoroacetate to select strains thatare resistant to relatively high levels of acetate and/or strains thatproduce relatively low levels of acetate.

A DNA sequence that encodes a polypeptide with one or more biochemicalactivities of the Zwf enzyme may be obtained by employing methods knownin the art (e.g., PCR amplification of a Zwf gene with suitable primersand cloning into a suitable DNA vector). The polynucleotide sequences ofsuitable Zwf genes have been disclosed. (See, e.g., GenBank). Forexample, the polynucleotide sequence of the A. succinogenes Zwf gene hasbeen published (SEQ ID NO:5 & 6). (See Joint Genome Institute,Department of Energy website). The E. coli Zwf gene is deposited withGenBank (e.g., under GenBank Accession Number NC_(—)000913 (SEQ ID NO:1)and GenBank Accession Number M55005 (SEQ ID NO:2)). The Zwf gene orvariants thereof may be obtained by PCR amplification of amicroorganism's genomic DNA with appropriate primers.

The DNA vector may be any suitable vector for expressing the gene in arecombinant microorganism. Suitable vectors include plasmids, artificialchromosomes (e.g., bacterial artificial chromosomes), and/or modifiedbacteriophages (e.g., phagemids). The vector may be designed to exist asan epigenetic element and/or the vector may be designed to recombinewith the genome of the microorganism.

The DNA molecule typically includes a promoter operationally linked to apolynucleotide that encodes a polypeptide having Zwf enzyme activity.The promoter may be endogenous or native to the microorganism from whichthe recombinant microorganism is derived, or heterologous to themicroorganism (i.e., derived from a source other than the recombinantmicroorganism). Furthermore, the promoter may be the native promoter fora selected Zwf gene or may be a promoter other than the native promoterfor a selected Zwf gene (i.e., a non-Zwf gene promoter). Where therecombinant microorganism is a strain of A. succinogenes, a suitableendogenous or native promoter is the A. succinogenes phosphoenolpyruvate(PEP) carboxykinase promoter (SEQ ID NO:4), deposited under GenBankaccession number AY308832, including nucleotides 1-258, or a variantthereof. The promoter may be operationally linked to the Zwf gene usingcloning methods that are known in the art. For example, the promoter andZwf gene may be amplified by PCR using primers that include compatiblerestriction enzyme recognition sites. The amplified promoter and genethen may be digested with the enzyme and cloned into an appropriatevector that includes a suitable multiple cloning site.

In addition, the DNA molecule may include a selectable marker. Theselectable marker may impart resistance to one or more antibioticagents. For example, selectable markers may include genes for ampicillinresistance, streptomycin resistance, kanamycin resistance, tetracyclineresistance, chloramphenicol resistance, sulfonamide resistance, orcombinations of these markers. Typically, the selectable marker isoperationally linked to a promoter that facilitates expression of themarker. Plasmids and other cloning vectors that include selectablemarkers are known in the art.

The DNA molecule typically is used to transform a host cell. Suitablehost cells include any cell that is useful for storing and/or producingthe DNA molecule.

Suitable host cells may include cells that expresses any gene present onthe DNA molecule. Suitable host cells also may include cells that arecapable of producing an organic acid in a fermentation process, such assuccinic acid at a concentration suitable for commercial production(e.g., at least about 20 g/L, more suitably at least about 50 g/L, andmore suitably at least about 100 g/L).

The methods for producing an organic acid typically include fermenting anutrient medium with a recombinant microorganism that expresses a Zwfgene. For example, the method may include fermenting a nutrient mediumwith a recombinant A. succinogenes that expresses a Zwf gene (e.g., aheterologous Zwf gene such as the E. coli Zwf gene). Organic acidsproduced in the fermentation may include succinic acid. One suitablerecombinant microorganism for the methods is a recombinant strain of A.succinogenes that expresses the E. coli Zwf gene, deposited under ATCCaccession number PTA-6255. The methods also may include fermenting anutrient medium with a recombinant strain of Bisgaard Taxon 6 orBisgaard Taxon 10 that express a Zwf gene (e.g., a heterologous Zwf genesuch as the E. coli Zwf gene) to produce succinic acid. The methods alsomay include fermenting a nutrient medium with a recombinant strain of E.coli that expresses a Zwf gene (or overexpresses a Zwf gene) to produceone or more organic acids such as lactic acid.

The methods may employ recombinant microorganisms that are resistant torelatively high levels of the organic acid being produced (e.g.,succinic acid). The methods also may employ strains of microorganismsthat are resistant to relatively high levels of undesirable by-productsand/or strains of microorganisms that produce relatively low levels ofundesirable by-products.

The nutrient medium typically includes a fermentable carbon source. Thefermentable carbon source may be provided by a fermentable biomass. Afermentable biomass may be derived from a variety of crops and/orfeedstocks including: sugar crops (e.g., sugar, beets, sweet sorghum,sugarcane, fodder beet); starch crops (e.g., grains such as corn, wheat,sorghum, barley, and tubers such as potatoes and sweet potatoes);cellulosic crops (e.g., corn stover, corn fiber, wheat straw, andforages such as Sudan grass forage, and sorghum). The biomass may betreated to facilitate release of fermentable carbon source (e.g.,sugars). For example, the biomass may be treated with enzymes such ascellulase and/or xylanase, to release simple sugars. The fermentablecarbon source may include simple sugars and sugar alcohols such asglucose, maltose, mannose, mannitol, sorbitol, galactose, xylose,arabinose, and mixtures thereof.

The methods typically result in a relatively high yield of succinic acidrelative to an input carbon source such as glucose. For example, themethods may have a succinic acid yield (g) of at least about 90%relative to glucose input (g). Alternatively, the yield may becalculated as % succinic acid yield (mol)/glucose input (mol). As such,the methods may have a succinic acid yield (mol) of at least about 140%relative to glucose input (mol). Desirably, the methods may have asuccinic acid yield (mol) of at least about 130% or at least about 170%relative to glucose input (mol).

The methods also typically result in a relatively high concentration ofsuccinic acid production (e.g., relative to a method that uses anon-recombinant microorganism in a fermentation). For example, afermentation may reach a concentration of at least about 50 g/L succinicacid. Desirably, a fermentation may reach a concentration of at leastabout 90 g/L succinic acid or more desirably, a concentration of atleast about 130 g/L succinic acid. In some embodiments, the fermentationtypically does not produce substantial levels of undesirable by-productssuch as acetate, formate, pyruvate, and mixtures thereof (e.g., no morethan about 2.0 g/L acetate, no more than about 2.0 g/L formate, and/orno more than about 3.0 g/L pyruvate).

The methods may be used to produce relatively high concentration oflactic acid (e.g., relative to a method that uses a non-recombinantmicroorganism in a fermentation). For example, the recombinantmicroorganisms may be used in a fermentation to produce lactic acid at aconcentration of at least about 25 g/L. In one embodiment, thefermentation yields may yield about 0.5 g lactic acid per gram glucose.The methods for producing lactic acid may include fermenting a suitablecarbon source with recombinant E. coli that expresses (or overexpresses)a polypeptide that has one or more biochemical activities of the Zwfgene. For example, the method may include fermenting a suitable carbonsource with recombinant E. coli that expresses the E. coli Zwf gene froman epigenetic element such as a plasmid.

Illustrated Embodiments

In one embodiment, the recombinant microorganism is a recombinant strainof Actinobacillus succinogenes that expresses a heterologous Zwf gene.The heterologous Zwf gene may be optimized for expression inActinobacillus succinogenes. The heterologous Zwf gene may encode an E.coli Zwf enzyme. The recombinant strain may include recombinantActinobacillus succinogenes deposited under ATCC Accession NumberPTA-6255. The recombinant strain may be capable of producing succinicacid at concentrations of about 50 g/L to about 130 g/L (e.g., in afermentation system that utilizes a suitable carbon source). Therecombinant strain may be resistant to levels of sodiummonofluoroacetate of at least about 1 g/L.

In some embodiments, the recombinant strain is a recombinant strain ofmicroorganism belonging to Bisgaard Taxon 6 or Bisgaard Taxon 10 thatexpresses a heterologous Zwf gene. The heterologous Zwf gene may encodeE. coli Zwf enzyme.

In another embodiment, the recombinant strain is a recombinant strain ofActinobacillus succinogenes, which includes a DNA molecule comprising atranscription promoter for Actinobacillus succinogenes operationallylinked to a heterologous Zwf gene. The transcription promoter mayinclude the A. succinogenes phosphoenolpyruvate (PEP) carboxykinasepromoter or a variant thereof (e.g., a polynucleotide of SEQ ID NO:4 ora polynucleotide having at least about 95% sequence identity to SEQ IDNO:4, where the polynucleotide has A. succinogenes phosphoenolpyruvate(PEP) carboxykinase promoter activity). The heterologous Zwf gene mayencode E. coli Zwischenferment enzyme or a variant thereof (e.g., apolynucleotide of SEQ ID NO:1 or a polynucleotide having at least about95% sequence identity to SEQ ID NO:1, where the polynucleotide has E.coli Zwischenferment enzyme activity). The heterologous Zwf gene mayinclude the E. coli Zwf gene. Optionally, the Zwf gene may be optimizedfor expression in Actinobacillus succinogenes. The DNA molecule may beepigenetic (e.g., present on a plasmid). The DNA molecule may include aselectable marker (e.g., kanamycin resistance, ampicillin resistance,streptomycin resistance, sulfonamide resistance, tetracyclineresistance, chloramphenicol resistance, or a combination thereof).

In another embodiment, the recombinant strain is a recombinant strain ofActinobacillus succinogenes which comprises a heterologous Zwf enzyme.The heterologous Zwf enzyme may be expressed from a Zwf gene that hasbeen optimized for expression in Actinobacillus succinogenes. Theheterologous Zwf enzyme may include E. coli Zwischenferment enzyme. Therecombinant strain may include recombinant A. succinogenes depositedunder ATCC Accession Number PTA-6255. The recombinant strain may becapable of producing succinic acid at concentrations of about 50 g/L toabout 130 g/L. Optionally, the recombinant strain is resistant to levelsof sodium monofluoroacetate of at least about 1 g/L.

In one embodiment, the method for producing succinic acid includesfermenting a nutrient medium with a recombinant microorganism thatexpresses a heterologous Zwf gene. The recombinant microorganism mayinclude a recombinant strain of Actinobacillus succinogenes (e.g., A.succinogenes recombinant strain deposited under ATCC Accession NumberPTA-6255). The recombinant microorganism may include a recombinantstrain of Bisgaard Taxon 6 or a recombinant strain of Bisgaard Taxon 10.The heterologous Zwf gene may include the E. coli Zwf gene. Optionally,the recombinant strain is resistant to levels of sodiummonofluoroacetate of at least about 1 g/L. Optionally, the recombinantstrain is capable of producing succinic acid at concentrations of about50 g/L to about 130 g/L. The nutrient medium may include a fermentablesugar (e.g., glucose). Typically, the method results in a succinic acidyield (g) of at least about 100% relative to glucose (g).

In one embodiment, the recombinant DNA molecule includes a transcriptionpromoter for A. succinogenes operationally linked to a heterologous Zwfgene. For example, the transcription promoter may include the A.succinogenes phosphoenolpyruvate (PEP) carboxykinase promoter or avariant thereof, (e.g., a polynucleotide of SEQ ID NO:4 or apolynucleotide having at least about 95% sequence identity to SEQ IDNO:4, where the polynucleotide has Actinobacillus succinogenesphosphoenolpyruvate (PEP) carboxykinase promoter activity).

In one embodiment, the recombinant DNA molecule is present in a DNAplasmid. Typically, the DNA plasmid includes a selectable marker (e.g.,a gene selected from the group consisting of ampicillin resistance,kanamycin resistance, streptomycin resistance, tetracycline resistance,chloramphenicol resistance, sulfonamide resistance, and combinationsthereof). The DNA molecule, which may be present in a DNA plasmid, maybe present in a host cell. The host cell may be capable of producingsuccinic acid at concentrations of about 50 g/L to about 130 g/L in afermentation system.

In one embodiment, the recombinant microorganism is a recombinant strainof a succinic acid producing microorganism which has been transformedwith a DNA molecule that expresses a polypeptide having Zwf enzymeactivity. The DNA molecule may include a polynucleotide that encodes apolypeptide having Zwf enzyme activity, which may include NADP reductaseactivity. The DNA molecule may include a polynucleotide that encodes apolypeptide having at least about 90% sequence identity (or desirably atleast about 95% sequence identity) to the amino acid sequence of a Zwfenzyme (e.g., SEQ ID NO:3 or SEQ ID NO:6), where the polypeptide has Zwfenzyme activity (e.g., NADP reductase activity). The DNA molecule mayinclude a polynucleotide sequence having at least about 90% sequenceidentity (or desirably at least about 95% sequence identity) to thepolynucleotide sequence of a Zwf gene (e.g., SEQ ID NO:1; SEQ ID NO:2;or SEQ ID NO:5), where the polynucleotide encodes a polypeptide havingZwf enzyme activity. In some embodiments, the recombinant strain may bederived from a microorganism whose 16S rRNA has at least about 90%sequence identity to 16S rRNA of Actinobacillus succinogenes. Forexample, the recombinant strain may be derived from a strain ofActinobacillus succinogenes, Bisgaard Taxon 6, or Bisgaard Taxon 10.

In another embodiment, the recombinant microorganism is a recombinantstrain of a succinic acid producing microorganism that has beentransformed with a heterologous Zwf gene. The heterologous Zwf gene maybe optimized for expression in the microorganism. In some embodiments,the heterologous Zwf gene may encode E. coli Zwf enzyme. In someembodiments, the Zwf gene may include a polynucleotide having at leastabout 95% sequence identity to SEQ ID NO:1, where the polynucleotide hasZwf enzyme activity.

In another embodiment, the recombinant microorganism is a recombinantstrain of a succinic acid producing microorganism that has beentransformed with a DNA molecule that includes a transcription promoterfor phosphoenolpyruvate (PEP) carboxykinase operationally linked topolynucleotide encoding a polypeptide having Zwf enzyme activity. Thetranscription promoter may include the Actinobacillus succinogenesphosphoenolpyruvate (PEP) carboxykinase promoter. In some embodiments,the promoter may include a polynucleotide having at least about 95%sequence identity to SEQ ID NO:4, where the polynucleotide has promoteractivity.

In another embodiment, the recombinant microorganism is a recombinantstrain transformed with a DNA molecule that is epigenetic. The DNAmolecule may be present on a plasmid.

In another embodiment, the recombinant microorganism is a recombinantstrain that is capable of producing succinic acid at concentrations ofabout 50 g/L to about 130 g/L.

The recombinant strain may be resistant to levels of sodiummonofluoroacetate of at least about 1 g/L. In some embodiments, therecombinant strain is recombinant Actinobacillus succinogenes depositedunder ATCC Accession Number PTA-6255.

In another embodiment, the recombinant microorganism is used forproducing succinic acid in a method that include fermenting a nutrientmedium with the recombinant microorganism. The nutrient medium typicallyincludes fermentable sugar such as glucose. The method may result in asuccinic acid yield (g) of at least about 100% relative to glucose (g).

In some embodiments, the DNA molecule comprising a transcriptionpromoter for a succinic acid producing microorganism operationallylinked to a heterologous Zwf gene. The transcription promoter mayinclude a phosphoenolpyruvate (PEP) carboxykinase promoter. In someembodiments, the promoter includes a polynucleotide having at leastabout 95% sequence identity to SEQ ID NO:4, where the polynucleotide haspromoter activity. The DNA molecule may be present within a plasmid. TheDNA molecule may be present in a host cell (e.g., a host cell capable ofproducing succinic acid at concentrations of about 50 g/L to about 130g/L).

EXAMPLES

Microorganism Strains and Plasmids

A. succinogenes strain FZ45 is a stable bacterial variant ofActinobacillus succinogenes 130Z, which is resistant to sodiummonofluoroacetate. See Guettler et al., INT'L J. SYST. BACT. (1999)49:207-216; and U.S. Pat. No. 5,573,931. The E. coli-A. succinogenesshuttle vector pLS88 (deposited at the American Type Culture Collectionas ATCC accession no. 86980) was obtained from Dr. Leslie Slaney,University of Manitoba, Canada. Plasmid pLS88 is described as havingbeen isolated from Haenzophilus ducreyi and may confer resistance tosulfonamides, streptomycin, and kanamycin.

Genetic Manipulations

Recombinant DNA manipulations generally followed methods described inthe art. Plasmid DNA was prepared by the alkaline lysis method. Typicalresuspension volumes for multicopy plasmids extracted from 1.5 mlcultures were 50 μl. Larger DNA preparation used the Qiagen PlasmidPurification Midi and Maxi kit according to the manufacturer'sinstructions. Restriction endonucleases, Molecular Weight Standards, andpre-stained markers were purchased from New England Biolabs andInvitrogen and digests were performed as recommended by themanufacturers, except that an approximately 5-fold excess of enzyme wasused. DNA was analyzed on Tris-acetate-agarose gels in the presence ofethidium bromide. DNA was extracted from agarose gels and purified usingthe Qiagen gel extraction kit according to the manufacturer'sinstructions. DNA was dephosphorylated using shrimp alkaline phosphatase(Roche) in combination with restriction digests. The phosphatase washeat inactivated at 70° C. for 15 min. Ligations were performed using a3- to 5-fold molar excess of insert to vector DNA in a 20 μl reactionvolume and 1 μl of T4 DNA Ligase (New England Biolabs) for 1 hour at 25°C. E. coli transformation were performed by using “library efficiencycompetent cells” purchased from Invitrogen, following the manufacturer'sinstructions.

Transformations using ligation mixes were plated without dilutions onstandard LB plates containing the appropriate antibiotic. PCRamplifications were carried out using the Perkin Elmer manual as aguideline. Primer designs were based on published sequences (as providedat the National Center for Biotechnology Information (NCBI) database).The primers included engineered restriction enzyme recognition sites.Primers were analyzed for dimer and hairpin formation and meltingtemperature using the Vector NTI program. All primers were ordered fromthe Michigan State Macromolecular Structure Facility. PCR amplificationswere carried out in an Eppendorf Gradient Master Cycler, or in a PerkinElmer Thermocycler. Starting annealing temperatures were determinedusing the Vector NTI program for each primer pair. Restriction enzymesfor digesting the amplified products were purchased from Invitrogen orNew England Biolabs.

Plasmid pJR762.55

The A. succinogenes phosphoenolpyruvate (PEP) carboxykinase promotersequence (P^(pepck), SEQ ID NO:4, GenBank accession number AY308832,including nucleotides 1-258) was amplified from A. succinogenes FZ45genomic DNA using the following primers: Forward,5′-AAAGAATTCTTAATTTCTTTAATCGGGAC (SEQ ID NO:7); and Reverse,5′-GCGTCGACATACTTCACCTCATTGAT (SEQ ID NO:8). EcoRI and SalI restrictionsequences (underlined nucleotides) were included to facilitate cloning,and the resulting 0.27-kb P^(pepck) fragment was inserted as anEcoRI/SalI fragment into pLS88 to produce plasmid pJR762.55.

Plasmid pJR762.73

The Zwf gene from E. coli was amplified from strain BL21(DE3) genomicDNA (ATCC accession number NC_(—)000913), using the following primers:Forward, 5′-CCGCTCGAGGGCGGTAACGCAAACAGC (SEQ ID NO:9); and Reverse,5′-CCGCTCGAGTTACTCAAACTCATTCCAGG (SEQ ID NO:10). XhoI restrictionsequences (underlined nucleotides) were included to facilitate cloningand the ensuing 1.5 kb Zwf fragment was inserted into the SalI site ofpJR762.55 to produce plasmid pJR762.73. Transformation of A.succinogenes

A. succinogenes competent cells for electroporation were prepared bygrowing cells in tryptic soy broth medium (TSB) to an OD₆₀₀ of ˜0.6.Cells were spun down, washed twice with sterile water, twice with 10%v/v glycerol and resuspended in 0.01× the original culture volume with10% glycerol. Cells were flash frozen and stored at minus 80° C.Approximately 40 μl of thawed cells were used for electroporation, in0.1 cm cuvettes with a BioRad GenePulser at settings of 400 W, 25 mF,and 1.8 kV. Following electroporation, 1 ml room temperature TSB mediumwas immediately added and the cells were incubated at 37° C. for 1 h.The cell solution was plated on TSB agar plates containing Kanamycin(100 μg/ml).

Optical Density Determination of A. succinogenes

Samples from magnesium-neutralized fermentations were spun at 420×g for2 min to precipitate the MgCO₃ and diluted with 0.5N HCl to solubilizeany remaining precipitate before reading at OD₆₆₀.

A. succinogenes Batch Fermentations

A. succinogenes fermentations were performed in 51 fermentors containingthe following medium unless otherwise specified: 80 g/L glucose, 85 g/Lliquid feed syrup (LFS), 0.2 mg/L biotin, 5 mM phosphate, 3 g/L yeastextract, Sensient AG900. The pH was maintained at 7.0 with a Mg(OH)₂.Agitation was set at 250 rpm, temperature at 38° C., and carbon dioxidewas sparged at a rate of 0.025 v.v.m. Fermentors were inoculated with a1.25% Seed inoculum, raised in serum vials containing the mediumdescribed above. The fermentation medium for recombinant strains of A.succinogenes contained 100 μg/ml Kanamycin.

Clearing of LFS

For fermentations that required a measure of growth through opticaldensity measurements a cold water extract of LFS was used. Suspendedsolids and some oils were removed through centrifugation of LFS in aSorvall GSA rotor, at 9,000 rpm for 20 minutes. The supernatant wasallowed to settle in a separation funnel for 3 hours at roomtemperature. The lower water phase typically represented 57% (w/v) ofthe raw LFS.

Biochemical Assays to Verify Zwf Expression

Glucose-6-phosphate dehydrogenase assays were performed as described byChoi et al., 2003. (See Choi, Jae-Chulk, Shin, Hyun-Dong, Lee, Yong-Hyun(2003) Enzyme and Microbial Technology 32, p. 178-185; “Modulation of3-hydroxyvalerate molar fraction onpoly(3-Ihydroxybutyrate-3-hydroxyvalerate) using Ralstonia eutrophatransformant co-amplifying phbC and NADPH generation-related Zwfgenes”). The formation rate of D-6-phospho-glucono-δ-lactone wasmeasured by the increase in NADPH, which was quantified by measuring theabsorbance at 340 nm. Each assay was performed in 1 ml containing, 50 μl[1M] Tris-HCl, pH 7.5, 200 μl [50 mM] MgCl₂, 100 μl [10 mM] NADP, 100 μl[10 mM] glucose-6-phosphate, 450 μl H2O, and 100 μl cell extract. Thespecific activity was calculated as: SpecificActivity=dA/dt/0.623×(protein concentration), or μmol/min mg⁻¹.Increased Zwf activity was observed in all recombinant strains thatinclude the plasmid pJR762.73, which expresses the E. coli Zwf gene fromthe A. succinogenes PEPCK promoter. Increased activity was observed intransformed Actinobacillus strain (FZ45) and in transformed strains ofBisgaard Taxon 6 (BT6) and Bisgaard Taxon 10 (BT10), which carried theplasmid pJR762.73. These results are illustrated in FIG. 3.

E. coli Fermentations

E. coli strains DH5α/pJR762.73 (Zwf), DH5α/pJR762.17 (Zwf), andDH5α/pLS88 were grown in NBS 5-liter Bioflo III fermentors using fourliters of the following medium: 900AG yeast extract 15 g; corn steepliquor 15 g; Na₂HPO₄ 1.16 g; NaH₂PO₄H₂O, 0.84 g: (NH₄)₂SO₄ 3 g;MgSO₄.7H₂O, 0.61; CaCl₂.2H₂O, 0.25 g, and glucose, 45 g per liter. ThepH was controlled at 6.7 through the automatic addition of K₂CO₃ (3.3N).The fermentations were each started with a 1.25% inoculum. Conditionswere initially made aerobic which favored the rapid growth of the E.coli cells; stirring was at 500 rpm and the medium was sparged with airat 0.5 liter/liter-min. Fermentor conditions were made anaerobic tofavor organic acid production when the cell density reached a minimum of6.2 OD₆₆₀ units; then the medium was sparged with 0.2 liter/liter-min ofa CO₂ and H₂ mixture (95:5), and stirring was reduced to 250 rpm.Samples were taken periodically and the organic acid products andresidual glucose concentrations were determined through HPLC.

Analysis of Fermentation Broths

Succinic acid, glucose, lactic acid, pyruvate, ethanol, and formic acidconcentrations were determined by reverse phase high pressure liquidchromatography (HPLC) using a Waters 1515 Isocratic pump with a Waters717 Auto sampler and a Waters 2414 refractive index detector set at 35°C. The HPLC system was controlled, data collected and processed usingWaters Breeze software (version 3.3). A Bio-Rad Aminex HPX-87H (300mm×7.8 mm) column was used with a cation H guard column held at 55° C.The mobile phase was 0.021 N sulfuric acid running at 0.5 ml/min.Samples were filter through a 0.45 μm filter, and 5.0 μl was injectedonto the column. Run time was thirty minutes.

CO₂ Measurements

A mass flow controller (Brooks model 58501) was used to monitor andsupply CO₂ to the fermentor sparging system at 100 ml/min. A mass flowmeter (Brooks model 58601) was used to measure CO₂ exiting the fermentorby way of the exhaust condenser system. The two CO₂ flow meters wereconnected to a computer via a 4-20 ma Bio-Command Interface. TheBioCommand Plus Bioprocessing software logged the inlet and outlet CO₂flow every 60 seconds. The rate of CO₂ consumption (ml/min) wasexpressed as the difference between the inlet and outlet rates duringany given minute (CO₂use=CO₂ in−CO₂out). The volume of CO₂ consumedduring any given fermentation interval is the sum of rates each minuteof the interval. The moles of CO₂ consumed were calculated using theIdeal Gas Law, (consumed liters/22.4 liters/mole=consumed moles).

The mass flow meters were calibrated by the manufacturer for CO₂ andtheir precision was 1% of full scale or 2 ml/m. The fermentation set-upwas monitored for gas leaks by mixing 5% hydrogen into the CO₂. Hydrogenleaks were detected using a Tif8800 CO/Combustible Gas analyzer.

Metabolic Flux Analysis of A. succinogenes Fermentations

The metabolic flux distributions (MFA) during anaerobic succinic acidproduction in Actinobacillus succinogenes were analyzed using theFluxAnalyzer software package. The FluxAnalyzer package was obtainedfrom Professor Steffen Klamt (Max Planck Institute, Magdeburg, Germany)and was operated according to the instructions provided in the manual.The FluxAnalyzer package facilitates the analysis of metabolic fluxes byproviding a graphical user interface for the MATLAB program, whichperforms the actual mathematical calculations. The MATLAB software waspurchased from MathWork Inc. By measuring the changes in extracellularconcentrations of the known and expected components of the entiremetabolic pathway, the intracellular fluxes for the major intracellularmetabolites were calculated using the metabolic network model describedbelow. The specific network (labeled A_succinogenes) was constructedusing the 20 known metabolites and 27 reactions shown below (withoutconsiderations of biomass composition and growth rate):

A_Succinogenes Metabolic Network Model

A_succinogenes Metabolic Network Model Glucose (in) → Glucose (R1)Glucose → Glucose-6P (R2) Glucose-6P + 2 NAD → 2 PEP + 2 NADH (R3) PEP →Pyruvate (R4) PEP + CO₂ → OAA (R5) Pyruvate → Pyruvate (out) (R6)Pyruvate + NAD → Acetyl-coA + NADH + CO₂ (R7) Pyruvate + NADH + CO₂ →Malic acid (R8) Acetyl-coA → Acetate (R9) Acetate → Acetate (out) (R10)Acetate + OAA → Citrate (R11) Citrate + NAD → CO₂ + NADH + α-KG (R12)OAA + NADH → Malic acid + NAD (R13) Malic acid → Fumarate (R14)Fumarate + NADH → Succinic acid + NAD (R15) Succinic acid → Succinicacid (out) (R16) CO₂ (in) → CO₂ (R17) Glycerol (in) → Glycerol (R18)Glycerol + 2 NAD → PEP + 2 NADH (R19) Sorbitol (in) → Sorbitol (R20)Sorbitol + NAD → Glucose-6P + NADH (R21) Xylose (in) → Xylose (R22)Xylose → R5P (R23) R5P + 5/3 NAD → 5/3 PEP + 5/3 NADH (R24) Glucose-6P +2 NADP → R5P + CO₂ + 2 NADPH (R25) Acetyl-coA + 2 NADH → Ethanol + 2 NAD(R26) Ethanol → Ethanol (out) (R27)

Fermentation samples were analyzed over the time course of thefermentations using the analytical methods previously described.Concentrations of glucose, glycerol, arabinose, xylose, sorbitol,succinic acid, acetic acid, ethanol, pyruvate, lactic acid andfermentation volumes were determined at each sampling time. The amountof metabolite was calculated according to the formula: (metabolite, g)=V(fermentor, 1)*C(metabolite, g/l). The metabolite consumption rate orthe metabolite production rate during the time period of t₀−t₁ wascalculated using the formula: Metabolite consumption rate (mol/h, t₀ andt₁)=[Amount (metabolite, g, t₀)−Amount (metabolite, g,t₁)]/[(t₁−t₀)*Molecular Weight of Metabolite]. For comparison ofmetabolic flux for all the time periods, the consumption rate orproduction rate of metabolite in the flux map was adjusted, assuming aglucose consumption rate in the flux map of 100. The metaboliteconsumption or production rate in the map “Mcp” was determined accordingto the following formula: Mcp=(metabolic consumption/or productionrate)×100/(glucose consumption rate).

The consumption or production rates of the various metabolites wereinput into the metabolic network model in the FluxAnalyzer packageaccording to the operating instructions. The function “Calculate/BalanceRates” was used to calculate all the calculatable rates. If the systemwas non-redundant, an optimization procedure was started, where a linearobjective function was minimized. If the system was redundant, one ormore of three methods (simple least squares, variances-weighted leastsquares I and variances weighted least squares II) were applied tocalculate the rates. The flux rate was shown directly on the flux map.Final flux map were copied into Microsoft Excel files for storagepurposes.

Metabolic Flux Analysis of Biochemical Pathways in A. succinogenes FZ45

Metabolic flux analysis was used to evaluate the effect of differentcarbon sources on succinic acid production in batch fermentations withA. succinogenes FZ45. The analyses established that the major pathwayfor succinic acid production in A. succinogenes FZ45 flows in thefollowing manner: phosphoenolpyruvate (PEP→*oxaloacetate(OAA)→malate→fumarate→succinic acid. The glyoxylate shunt and thePEP-transport-system (PTS) appear not to be substantially used in theorganism. Glucose fermentations reach a concentration of 61.7 g/Lsuccinic acid with a yield of about 94% (succinic acid (g)/glucose (g)).Fermentations performed using a more reduced carbon source, such assorbitol, produced higher amounts of succinic acid (77.3 g/L) with ahigher yield (108% succinic acid (g)/glucose (g)), indicating thatreducing power may become a limiting factor during the fermentation ofglucose.

Enhanced Succinic Acid Production from Glucose by Over-Expression of Zwf

Strains FZ45, FZ45/pLS88 and FZ45/pJR762.73 were cultured under standardproduction conditions with the exception that 100 μg/ml Kanamycin wasadded to the fermentation medium for the transformed strains. FZ45/pLS88served as a second control, and is transformed with the cloning vector,carrying no PEP carboxykinase promoter or Zwf gene. The carbon sourceused was glucose. The strain FZ45/pJR762.73 showed an increase insuccinic acid production over the control strains FZ45 and FZ45/pLS88,with a corresponding increase in the final concentration of succinicacid. The total amount of succinic acid produced from glucose increasedfrom 284 g to 302 g, the molar yield of succinic acid produced increasedfrom 144% to 155% (moles succinic acid/100 moles glucose), the weightyield increased from 94.7% to 101.9%, and the final concentration ofsuccinic acid in the fermentation broth increased from 62 g/L to 65 g/L.These results are summarized in Table 1. All transformed FZ45derivatives exhibit slower growth compared to the untransformed FZ45,which may be caused by the replication of the additionalextrachromosomal plasmid DNA.

TABLE 1 Production of Succinic acid From Glucose by Strains FZ45,FZ45/pLS88 and FZ45/pJR762.73 Molar Weight Total Succinic acid Strainyield (%) yield (%) g/L [g] FZ45 144 94.7 61.7 284 FZ45/pLS88 149 98.060.4 272 FZ45/pJR762.73 155 101.9 65.4 302

Further, the strain FZ45/pJR762.73 also produced less of the twometabolites acetic acid and pyruvic acid, as shown in Table 2.

TABLE 2 Production of Other Metabolites by Strains FZ45 andFZ45/pJR762.73 Succinic Acid Pyruvic Acid Acetic Acid Strain [g/l] [g/l][g/l] FZ45 61.7 3.7 1.5 FZ45/pLS88 60.4 2.1 1.4 FZ45/pJR762.73 65.4 2.71.4

Metabolic flux analyses on both FZ45 and FZ45/pJR762.73 showed thatFZ45/pJR762.73 channeled more carbon into the pentose phosphate pathwaythan the untransformed FZ45 (See FIG. 1 and FIG. 2). Thus,over-expression of the Zwf protein was sufficient to enhance succinicacid yields and to reduce the production of other metabolites whenglucose was used as carbon source.

Fermentation with A. succinogenes FZ45/pJR762.73 Using a Reduced CarbonSource

Fermentations with A. succinogenes FZ45/pJR762.73 utilizing mannitol asa carbon source were also performed. Mannitol is a 6-carbonsugar-alcohol that is more reduced than glucose. Expression of Zwf alsoenhanced succinic acid production using mannitol (See Table 3). However,fermentations using this sugar alcohol also showed increased yields evenwith the untransformed strain FZ45. This indicates that increasing theamount of metabolic reducing equivalents will enhance succinic acidproduction.

TABLE 3 Production of Succinic Acid Using Mannitol as Carbon SourceSuccinic Carbon Molar Weight Acid Strain Source yield (%) yield (%) g/L[total g] FZ45 glucose 144 94.7 61.7 284 FZ45 mannitol 179 116.0 85 406FZ45/pJR762.73 mannitol 193 125.4 88 421Effect of Zwf Expression in Recombinant Bisgaard Taxon 6 and BisgaardTaxon 10

The effect of Zwf expression in other species was also tested using theorganisms Bisgaard Taxon 6 (BT6) and Bisgaard Taxon 10 (BT10). Bothorganisms belong to the family Pasteurellaceae, and are related to A.succinogenes. Also, both organisms are known to produce succinic acid.Using the methods described above and the same plasmid, pJR762.73(carrying the Zwf gene under the A. succinogenes PEPCK promoter),Bisgaard Taxa were transformed. Both these transformed strains showed anincrease in succinic acid production using glucose as the carbon source.These results are shown in Table 4 below.

TABLE 4 Production of Succinic acid from Glucose by StrainsBT6/pJR762.73 and BT10/pJR762.73 Molar Weight Succinic Acid Strain yield(%) yield (%) g/L [total g] BT6/pLS88 92 60.3 40 174 BT6/pJR762.73 9662.8 39 180 BT10/pLS888 132 86.5 56 255 BT10/pJR762.73 136 89.0 57 258

Flux analysis of these fermentations with the Bisgaard Taxa strainsindicated that use of the pentose phosphate pathway was indeed increasedin the strains carrying the plasmid. BT6/pJR762.73 routed more carbonthrough the pentose phosphate pathway than the control (33 mol % vs. 20mol %). Similarly, BT10/pJR762.73 routed 35 mol % carbon through thepentose phosphate pathway, compared to only 5 mol % in the control.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenillustrated by specific embodiments and optional features, modificationand/or variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numerical valuesare provided for embodiments, additional embodiments are described bytaking any 2 different values as the endpoints of a range. Such rangesare also within the scope of the described invention.

All references, patents, and/or applications cited in the specificationare incorporated by reference in their entireties, including any tablesand figures, to the same extent as if each reference had beenincorporated by reference in its entirety individually.

The invention claimed is:
 1. A genetically-modified microorganism of the family Pasteurellaceae that is transformed with at least one polynucleotide sequence encoding a glucose-6-phosphate dehydrogenase enzyme that is operably linked to a promoter sequence, wherein said microorganism produces an increased amount of succinic acid compared to a parent microorganism that is not transformed with the at least one polynucleotide sequence.
 2. A genetically-modified microorganism according to claim 1, wherein said microorganism comprises a 16S ribosomal RNA sequence with at least 90% sequence identity to the 16S ribosomal RNA sequence of Actinobacillus succinogenes.
 3. A genetically-modified microorganism according to claim 2, wherein said polynucleotide encodes an Esherichia coli glucose-6-phosphate dehydrogenase enzyme.
 4. A genetically-modified microorganism according to claim 2, wherein said promoter sequence comprises a phosphoenolpyruvate carboxykinase promoter operably linked to said polynucleotide encoding the glucose-6-phosphate dehydrogenase enzyme.
 5. A genetically-modified microorganism according to claim 4, wherein the microorganism is capable of producing succinic acid at concentrations of about 50 g/L to 130 g/L.
 6. A genetically-modified microorganism according to claim 2, wherein said polynucleotide encodes at least one Actinobacillus succinogenes glucose-6-phosphate dehydrogenase enzyme.
 7. A genetically-modified microorganism according to claim 6, which comprises a phosphoenolpyruvate carboxykinase promoter operably linked to said polynucleotide encoding an Actinobacillus succinogenes glucose-6-phosphate dehydrogenase enzyme.
 8. A genetically-modified microorganism according to claim 7, wherein said promoter is an Actinobacillus succinogenes promoter.
 9. A genetically-modified microorganism according to claim 8, wherein the microorganism is capable of producing succinic acid at concentrations of about 50 g/L to 130 g/L.
 10. A genetically-modified microorganism according to claim 1, wherein the microorganism is capable of producing succinic acid at concentrations of about 50 g/L to 130 g/L.
 11. A genetically-modified microorganism according to claim 2, wherein the polynucleotide is a variant having at least 90 percent sequence identity to a glucose-6-phosphate dehydrogenase gene.
 12. A genetically-modified microorganism according to claim 11, wherein the polynucleotide variant enzyme has at least 95 percent sequence identity to a glucose-6-phosphate dehydrogenase gene.
 13. A method of producing succinic acid, comprising culturing a genetically-modified microorganism according to claim 1 under conditions sufficient to produce succinic acid.
 14. A method of producing succinic acid comprising culturing a genetically-modified microorganism according to claim 2, under conditions sufficient to produce succinic acid.
 15. A genetically-modified microorganism according to claim 1, wherein said promoter sequence is operably linked to a polynucleotide encoding an endogenous glucose-6-phosphate dehydrogenase enzyme and wherein said promoter sequence and said polynucleotide are integrated into the genome of the microorganism.
 16. A method of producing succinic acid comprising culturing a genetically-modified microorganism according to claim 4 under conditions sufficient to produce succinic acid at concentrations of about 50 g/L to 130 g/L. 